The present invention relates generally to methods and formulations for the separation of biological macromolecules, and in particular, to the separation to single-stranded DNA fragments according to their size using capillary electrophoresis with improved polyacrylamide matrixes.
Capillary electrophoresis (CE) has demonstrated its advantages over standard slab gel based electrophoretic techniques as a rapid, high-throughput and high-resolution method for separation of biological macromolecules, such as proteins, peptides and nucleic acids (see e.g. Slater et al., Electrophoresis, 1998. 19(10), 1525-1541.) Capillary gel electrophoresis (CGE) is the CE-analog of traditional slab-gel electrophoresis and is most often used for size-based separation of biological macromolecules such as oligonucleotides, DNA restriction fragments and proteins. The separation is performed by filling the capillary with a sieving matrix, for example, cross-linked polyacrylamide, agarose or linear polymer solutions. The main advantages over slab-gel electrophoresis are a wider range of gel matrixes and compositions, on-line detection, improved quantitation and automation.
One of the major technological improvements in CE for DNA analysis was the introduction of replaceable polymer solutions as sieving matrixes (see, e.g., Sudor et al. Electrophoresis, 1991. 12:1056-1058; Ruiz-Martinez et al., Analytical Chemistry, 1993. 65(20):2851-2858.) The use of solutions of non-cross-linked synthetic or natural polymers has significantly increased the number of analyses per capillary column. Replacing the matrixes after each separation run creates reproducible analytical conditions. Another advantage of non cross-linked polymers is that they can be polymerized under controlled conditions in aqueous solutions, characterized, purified and stored indefinitely as dry powder (see, e.g., Goetzinger et al., Electrophoresis, 1998. 19(2):242-248.)
Solutions of polymers may then be prepared using various background electrolyte systems according to the requirements of a particular application. The versatility gained by the use of such entangled polymer solutions allows for a wide range of electrophoresis conditions to be utilized, such as elevated capillary temperature, without damaging the matrix structure (see, e.g., Kleparnik et al., Electrophoresis, 1996. 17(12):1860-1866). In contrast cross-linked gels are usually polymerized in situ under less than ideal conditions. Typically, these polymers are not fully characterized, and their quality varies from run-to-run. Cross-linked gel filled capillaries are also very prone to bubble formation due to temperature or osmotic stress. Additionally, replacement requires arduous removal, scrupulous clean up of the old material.
DNA separation applications (e.g. DNA sequencing, short tandem repeat [STR] analysis, differential display) traditionally performed using cross-linked polyacrylamide slab gels require both high resolving power (1 base resolution up to at least 500 bases) and strong denaturing capacity. This also applies for analogous applications using CE with replaceable polymer matrixes. Although replaceable linear polyacrylamide has been shown to provide unsurpassed separation efficiencies (see, e.g., Pariat et al., Journal of Chromatography, 1993. 652(1):57-66), it also exhibits sequence-specific anomalous migration of both double-stranded and single-stranded DNA (see, e.g., Berka et al., Electrophoresis, 1995. 16(3):377-388 and Wenz, Nucleic Acids Research, 1994. 22(19):4002-4008). Even under denaturing conditions (typically 6-8 M urea in the running buffer) anomalous migration of specific ssDNA fragments poses a serious problem causing compressions in DNA sequencing and low accuracy of DNA fragment sizing in genotyping applications. Thus, the secondary structure of single stranded DNA fragments can adversely affect the migration of DNA fragments in polymer solutions resulting in inaccurate sizing. In the area of DNA sequencing, the same secondary interaction of DNA fragments results in compressions that do not allow proper identification of the fragment sequence.
This invention discloses methods of biological macromolecule separation and formulations for linear polymer separation matrixes exhibiting improved DNA denaturation properties for use in capillary array electrophoresis. In a preferred embodiment, a combination of urea with organic denaturants (preferably 2-pyrrolidinone, N,Nxe2x80x2-dimethylacetamide, and N,Nxe2x80x2-dimethylformamide) resulted in a chemical environment strongly disfavoring base pairing of nucleic acids. Separation of single-stranded DNA fragments in these matrixes minimizes effects of DNA strand secondary structures on fragment mobility""s, thus greatly improving the size-dependent separation. Further, this invention discloses optimized background electrolyte concentration and run voltage to further improve size-dependent migration of DNA fragments, in addition to enhanced denaturing properties of polymer matrixes. Biological applications of capillary electrophoresis such as differential display of mRNA, dideoxyfingerprinting, STR sizing and DNA sequencing would strongly benefit from using such formulations.